Modified oligonucleotides, their preparation and their use

Abstract

The invention relates to novel modified oligonucleotides which contain at least one 8-azapurine base and form more stable hybridization complexes with nucleic acids; To a process for their preparation, and to their use as inhibitors of gene expression, as probes for detecting nucleic acids, as aids in molecular biology, and as a pharmaceutical or diagnostic agent.

We claim: 
     
       1. An oligonucleotide of the formula I ##STR17## and the physiologically tolerated salts thereof, in which R 1  is hydrogen, C 1  -C 18  -alkyl, C 2  -C 18  -alkenyl, C 2  -C 18  -alkynyl, C 2  -C 18  -alkylcarbonyl, C 3  -C 19  -alkenylcarbonyl, C 3  -C 19  -alkynylcarbonyl, (C 6  -C 14 )-aryl-(C 1  -C 8 )-alkyl, a protective group which is customary in nucleotide chemistry, or a radical of the formula IIa ##STR18## R 1a  is hydrogen, C 1  -C 18  -alkyl, C 2  -C 18  -alkenyl, C 2  -C 18  -alkynyl, C 2  -C 18  -alkylcarbonyl, C 3  -C 19  -alkenylcarbonyl, C 3  -C 19  -alkynylcarbonyl, (C 6  -C 14 )-aryl-(C 1  -C 8 )-alkyl, or a radical of the formula IIb ##STR19## R 2  is hydrogen, hydroxyl, C 1  -C 18  -alkoxy, C 1  -C 6  -alkenyloxy, halogen, azido or NH 2  ; a is oxy or methylene;   n is an integer from 3 to 99;   W is oxo, thioxo or selenoxo;   V is oxy, sulfanediyl or imino;   Y is oxy, sulfanediyl, imino or methylene;   Y' is oxy, sulfanediyl, imino, (CH 2 ) m  or V(CH 2 ) m'   in which   m is an integer from 1 to 18;   X is hydroxyl or mercapto;   U is hydroxyl, mercapto, SeH, C 1  -C 18  -alkoxy, C 1  -C 18  -alkyl, C 6  -C 20  -aryl, (C 6  -C 14 )-aryl-(C 1  -C 8 )-alkyl, NHR 3 , NR 3  R 4  or a radical of the formula III   (OCH.sub.2 CH.sub.2).sub.p O(CH.sub.2).sub.q CH.sub.2 R.sub.5(III)     in which     R 3  is C 1  -C 18  -alkyl, C 6  -C 20  -aryl, (C 6  -C 14 )-aryl-(C 1  -C 8 )-alkyl, or 2-(CH 2 ) c  -(NH(CH 2 ) c ) d  --NR 6  R 6 , in which c is an integer from 2 to 6 and d is an integer from 0 to 6, and R 6  are, independently of each other, hydrogen or C 1  -C 6  -alkyl or C 1  -C 4  -alkoxy-C 1  -C 6  -alkyl;   R 4  is C 1  -C 18  -alkyl, C 6  -C 20  -aryl or (C 6  -C 10 )-aryl-(C 1  -C 8 )-alkyl, or, in the case of NR 3  R 4 , is, together with R 3  and the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can additionally contain a further heteroatom selected from O, S and N,   p is an integer from 1 to 100,   q is an integer from 0 to 22,   R 5  is hydrogen or a functional group;   Z and Z' are, independently of each other, hydroxyl, mercapto, SeH, C 1  -C 22  -alkoxy, --O--(CH 2 ) b  --NR 6  R 7 , in which b is an integer from 1 to 6, and R 7  is C 1  -C 6  -alkyl, or R 6  and R 7 , together with the nitrogen atom carrying them, form a 3-6-membered ring, C 1  -C 18  -alkyl, C 6  -C 20  -aryl, (C 6  -C 14 )-aryl(C 1  -C 8 )-alkyl, (C 6  -C 14 )-aryl-(C 1  -C 8 )-alkoxy, where aryl is also heteroaryl, and aryl is optionally substituted by 1, 2, or 3 identical or different radicals from the group carboxyl, amino, nitro, C 1  -C 4  -alkylamino, C 1  -C 6  -alkoxy, hydroxyl, halogen and cyano, C 1  -C 18  -alkylmercapto, NHR 3 , NR 3  R 4 , a radical of the formula III or a group which favors intracellular uptake or which serves as the label of A DNA probe, or, when the olionucleotide analog hybridizes to the target nucleic acid, attacks the latter with binding, crosslinking or cleavage,and the curved bracket indicates that R 2  and the adjacent radical Y or Y' can be located either in the 2' and 3' positions or else, conversely, in the 3' and 2' positions, where each nucleotide can be present in its D or L configuration and the base B can be located in the α or β position, where   B are, independently of each other, a base which is customary in nucleotide chemistry, where at least one B is a base of the formula IV ##STR20## in which E and F are, independently of each other, H, OH, or NH 2 .   
     
     
       2. An oligonucleotide as claimed in claim 1, wherein the base B is located in the β position, the nucleotides are in the D configuration, R 2  is located in the 2' position and a is oxy. 
     
     
       3. An oligonucleotide as claimed in claim 1, wherein R 1  is hydrogen, C 1  -C 6  -alkyl, or a radical of the formula IIa;   R 1a  is hydrogen, C 1  -C 6  -alkyl, or a radical of the formula IIb;   R 2  is hydrogen, C 1  -C 6  -alkoxy, C 1  -C 6  -alkenyloxy, or hydroxyl;   n is an integer from 4 to 39;   m is an integer from 1 to 6;   U is hydroxyl, mercapto, C 1  -C 6  -alkoxy, C 1  -C 6  -alkyl, NR 3  R 4  or NHR 3 , in which   R 3  is C 1  -C 8  -alkyl or methoxyethyl, and   B, W, V, Y, Y', X and Z have the meanings listed in claim 1.   
     
     
       4. An oligonucleotide as claimed in claim 1, wherein V, Y, and Y' are each independently oxy, sulfanediyl or imino. 
     
     
       5. An oligonucleotide as claimed in claim 1, wherein W is oxo or thioxo. 
     
     
       6. An oligonucleotide as claimed in claim 1, wherein U is hydroxyl, methyl or mercapto. 
     
     
       7. An oligonucleotide as claimed in claim 1, wherein at least one of R 1  and R 1a  is hydrogen. 
     
     
       8. A process for preparing an oligonucleotide of the formula (I), or a physiologically tolerated salt thereof, as claimed in claim 1, said process comprising condensing nucleotides, in each case containing a nucleotide base, one at a time, onto an appropriately derivatized support or onto a growing oligomer chain. 
     
     
       9. A process for preparing a pharmaceutical or a diagnostic agent, said process comprising mixing at least one oligonucleotide as claimed in claim 1 with a physiologically acceptable excipient and also, and optionally, with suitable aditives and/or auxiliary substances. 
     
     
       10. A pharmaceutical or a diagnostic agent, containing at least one oligonucleotide as claimed in claim 1, and optionally together with a physiologically acceptable excipient and/or auxiliary substances. 
     
     
       11. An oligonucleotide as claimed in claim 1, wherein R 2  is C 1  -C 6  alkenyloxy. 
     
     
       12. An oligonucleotide as claimed in claim 11, wherein R 2  is C 1  -C 6  allyloxy. 
     
     
       13. An oligonucleotide as claimed in claim 1, wherein R 5  is a functional group and is selected from hydroxyl, amino, C 1  -C 18  -alkylamino, COOH, CONH 2 , COO(C 1  -C 4 )-alkyl and halogen. 
     
     
       14. An oligonucleotide as claimed in claim 1, wherein said bases customary in nucleotide chemistry are natural bases or unnatural bases or their prodrug forms. 
     
     
       15. An oligonucleotide as claimed in claim 14, wherein said natural bases are adenine, cytosine, thymine, guanine, uracil or hypoxanthine. 
     
     
       16. An oligonucleotide as claimed in clain 14, wherein said unnatural bases are purine, 8-azapurine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N 4  N 4  -ethanecytosine, N 6  N 6  -ethano-2,6-diaminopurine, pseudoisocytosine, 5-methylcytosine, 5-fluorouracil, 5-(C 3  -C 6 )-alkynyluracil, or 5-(C 3  -C 6 )-alkynylcytosine. 
     
     
       17. An oligonucleotide as claimed in claim 3, wherein R 1  is C 1  -C 6  -alkyl;   R 1a  is C 1  -C 6  -alkyl;   R 2  is C 1  -C 6  -alkenyloxy or hydrogen;   n is an integer from 5 to 29;   m is 1;   U is hydroxyl or C 1  -C 6  -alkyl,   R 3  is C 1  -C 4  -alkyl.   
     
     
       18. An oligonucleotide as claimed in claim 17, wherein R 1  is methyl;   R 1a  is methyl;   R 2  is C 1  -C 6  allyloxy or hydrogen.   
     
     
       19. An oligonucleotide as claimed in claim 17, wherein R 2  is hydrogen.